Enzymes using metal centers and/or organic radicals play many crucial roles in the fundamental biochemistry of human health, with deficiencies in their bioassembly or enzymatic functions associated with various diseases. The R. David Britt laboratory is using advanced spectroscopic techniques such as multifrequency electron paramagnetic resonance (EPR) to understand the assembly and catalytic mechanism of a number of such metal and radical centers. Many important enzymes involved in multielectron oxidation or reduction reactions employ metal clusters in their catalysis. The Britt laboratory is studying how such clusters are assembled by identifying and interrogating assembly intermediates with their spectroscopic methods. For example the [Fe-Fe] hydrogenase enzyme uses a complex multinuclear Fe-S ?H-cluster?, containing organometallic Fe-CO and Fe-CN components, to catalyze reversible interconversion of H2 with protons and electrons. How does nature safely assemble such a center involving potentially dangerous CO and CN- species? The Britt laboratory is exploring how a radical reaction catalyzed by HydG, a member of the ?radical SAM? superfamily of enzymes, safely forms Fe(CO)x(CN)y organometallic synthons at the earliest stage of cluster synthesis and how these are processed by other maturase enzymes in the synthesis of the catalytic cluster. Magnetic nuclear isotopes (e.g. 13C, 15N, 57Fe) provide important magnetic interactions with paramagnetic forms of such clusters, as observed by EPR-based methods such as electron-nuclear double resonance (ENDOR). These and other nuclei provide handles for other spectroscopic probes of FeS cluster assembly such as Mssbauer, NMR, and FTIR. Their ability to assemble the active site enzymatically via cell-free synthesis allows the Britt laboratory to isotope-edit the assembled H-cluster and probe intermediates in hydrogen catalysis with isotope specific spectroscopies. Parallel experiments are unraveling the biosynthesis of the complex Fe-S ?M-cluster? at the heart of the nitrogenase enzyme, which can incorporate Mo or V or an additional Fe in its active site. The Britt lab is studying other members of the radical SAM superfamily that can carry out a wide variety of reactions such as organic cofactor and vitamin biosynthesis and post translational modifications of short peptides. Further investigations with EPR and other spectroscopies will probe metalloenzymes and radical enzymes with diverse functions, including NO and Ca2+ signaling, organohalide detoxification, metal ion sequestration and homeostasis, and substrate oxygen insertion. In such projects the Britt laboratory works closely with a wide variety of collaborators working on numerous NIH-supported research projects. This supplement is designed to dramatically improve our EPR experimental capabilities. We have built a unique high frequency (263 GHz) pulse EPR spectrometer that will give us new capabilities for studying metal clusters, particularly integer spin states with large zero field splitting. Additionally, this high frequency instrument will provide high resolution orientation selective ENDOR on narrow line organic radical intermediates. Also, certain high spin half-integer spin metals such as S=5/2 Mn(II) exhibit dramatic line narrowing and hence enhanced EPR/ENDOR sensitivity at such high frequency and corresponding magnetic field. This supplement is designed to improve the capability of the first implementation of this instrument (funded by the NSF MRI program) in order to increase pulse power, add better stability and signal to noise, and improve signal processing capabilities, in order to make this instrument a superior high frequency resource targeting the relatively dilute enzyme intermediates that are the focus of our NIH R35 EPR studies.